Activation of the Wnt signaling pathway requires binding of extracellular Wnt ligands to the Frizzled receptor and to the co-receptor LRP5 (Accession number: UniProtKB-075197/LRP5_HUMAN) or its closely related homologue LRP6 (Accession number: UniProtKB-O75581/LRP6_HUMAN). There are 19 Wnt proteins and 10 Frizzled receptors in mammalian cells. In the absence of Wnt ligand, cytoplasmic beta-catenin is phosphorylated by a protein complex consisting of the scaffolding proteins Axin and APC and the kinases GSK3beta and CK1a. Subsequent recognition by the ubiquitin ligase beta-TrcP leads to ubiquitin-mediated degradation of beta-catenin. In the presence of Wnt ligand, binding of Wnt to Frizzled and LRP5 or LRP6 leads to recruitment of the cytoplasmic effector protein Dvl and phosphorylation of the LRP5 or LRP6 cytoplasmic tail, which provides the docking site for Axin. Axin sequestration by LRP5 or LRP6 leads to the inactivation of the Axin-APC-GSK3beta complex and, therefore, intracellular beta-catenin stabilization and accumulation. Hence, cytoplasmic levels of beta-catenin rise, and beta-catenin migrates to the nucleus and complexes with members of the T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF) family of transcription factors. Basal transcription machinery and transcriptional co-activators are then recruited, including cAMP response element-binding protein (CREB)-binding protein (CBP) or its homolog p300, leading to expression of various target genes, including Axin2, cyclin D1 and c-Myc.
An additional level of ligand-dependent Wnt pathway regulation is mediated by the E3 ligase RNF43, and its closely related homologue ZNRF3, and by the secreted R-Spondin proteins (de Lau et al. “The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength”. Genes Dev. 2014; 28(4):305-16). RNF43 mediates the ubiquitination of the Frizzled/LRP5 or LRP6 receptor complex at the cell surface, leading to its degradation and, thereby, inhibiting ligand-dependent Wnt pathway activity. The activity of RNF43 is counteracted by the R spondin family members (R-spondin 1 to 4 ligands). When R-Spondin ligand is present, it removes RNF43 from the cell surface, allowing Frizzled/LRP5 or LRP6 complex accumulation and enhancement of Wnt signaling in the presence of Wnt ligands.
LRP5 and LRP6 function as gatekeepers of ligand dependent Wnt signaling activation and, therefore, may be considered as targets to achieve complete blockade of the pathway mediated by all 19 Wnt ligands and 10 Frizzled receptors and enhanced by R-spondin ligands. In particular, Wnt ligands can be divided into a Wnt1 class and a Wnt3a class, each binding to different epitopes/regions of LRP5 and LRP6 for signaling. The ectodomain of LRP5 and LRP6 comprises four repeating units of a beta-propeller connected to an EGF-like domain, followed by three LDLR-type A repeats. Combined structural and functional analyses of LRP5 and LRP6 suggest that Wnt1 (Wnt1-class ligand) binds to a fragment containing beta-propeller 1 and 2 and Wnt3a binds to a fragment containing beta-propeller 3 and 4 of LRP6. So far, only a low-resolution picture of LRP6 ectodomain containing beta-propeller from 1 to 4 regions is reported (Ahn et al. “Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6”. Dev Cell. 2011; 21(5):862-73). However, the uncertainties of these low-resolution reconstructions (40 A°) and the absence of structural data of the LRP6 ectodomain in complex with the Wnt ligands do not allow defining the exact epitopes involved in Wnt1 or Wnt3a ligand binding.
Hyperactivation of Wnt signaling is involved in the pathogenesis of various types of cancer. In some cancer types frequent mutations in downstream signaling molecules contribute to constitutively activated Wnt pathway (e.g. APC mutations in colorectal cancer; beta-catenin activating mutation in hepatocellular carcinoma). In contrast, in Triple Negative Breast Cancer (TNBC), Non Small Cell Lung Cancer (NSCLC), pancreatic adenocarcinoma and in a subset of Colo-Rectal Cancer (CRC) and endometrial cancers, Wnt signaling activation is driven by a ligand dependent mechanism (i.e. by an autocrine/paracrine Wnt activation), as detected by beta-catenin intracellular accumulation. In NSCLC, TNBC and pancreatic adenocarcinoma, ligand dependent Wnt activation is mediated by multiple mechanisms, including increased expression of the Wnt ligands and/or of LRP5 and LRP6 receptors, or silencing of LRP5 and LRP6 negative regulator DKK1 (TNBC: Liu et al. “LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy”. Proc Natl Acad Sci USA 2010; 107 (11):5136-41; Khramtsov et al. “Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome”. Am J Pathol. 2010; 176(6): 2911-20; NSCLC: Nakashima et al. “Wnt1 overexpression associated with tumor proliferation and a poor prognosis in non-small cell lung cancer patients”. Oncol Rep. 2008; 19(1):203-9; Pancreatic cancer: Zhang et al. “Canonical wnt signaling is required for pancreatic carcinogenesis”. Cancer Res. 2013; 73(15):4909-22). In particular, published data have shown that in healthy tissues (e.g. mammary and lung epithelium), beta-catenin is localized solely at the plasma membrane. In contrast, the majority of TNBC, NSCLC and pancreatic adenocarcinoma primary clinical samples showed beta-catenin intracellular accumulation (i.e. in the cytoplasm/nucleus; biomarker of Wnt signaling activation), due to aberrant Wnt signaling. Recent publications have shown that ligand dependent Wnt signaling activation is mediated by mutated/inactivated RNF43 (Giannakis et al. “RNF43 is frequently mutated in colorectal and endometrial cancers”. Nat Genet. 2014; 46(12):1264-6) or by activating R-Spondin fusion transcripts (encoding R-spondin2 or R-spondin3 proteins driven by constitutively active strong promoters; Seshagiri et al. “Recurrent R-spondin fusions in colon cancer”. Nature 2012; 488(7413):660-4) in a subset of CRC and endometrial cancers. Inactivating RNF43 mutations and R-Spondin fusion transcripts have both been shown to augment ligand dependent Wnt signaling in vitro by increasing the abundance of Frizzled on the cell surface. Ligand dependent Wnt activation in tumors was shown to drive tumor growth and resistance to chemotherapy or immunotherapy, and is linked to recurrence in pre-clinical models.
Some LRP5 or LRP6 binding molecules, able to modulate the Wnt signaling pathway, are known in the art:
Dickkopf-1 (DKK1) is a LRP5 and LRP6 inhibitor. DKK1 associates with both the Wnt co-receptors, LRP5 and 6, and the transmembrane protein, Kremen, inhibits Wnt signaling and leads to rapid LRP5 and LRP6 internalization. It is shown that DKK1 inhibits both Wnt1 and Wnt3a mediated signaling. Structural modeling studies show that a single DKK1 molecule cooperatively binds to an extended region of the LRP6 ectodomain (from beta-propeller 1 to 3). The structural analyses suggest a DKK1 cooperative binding-interaction with LRP6 with an initial binding to the beta-propeller 3 region that facilitates the interaction/binding to the beta-propeller 1 and 2 region via a conformation change of the LRP6 ectodomain. However, elucidation of the defined epitopes within the beta-propeller 1, 2 and 3 domains involved in the DKK1 binding to LRP6 is lacking due to the low resolution of the structural reconstructions of the full LRP6 ectodomain bound to DKK1, as mentioned.
It was shown that DKK1 treatment in vivo causes severe toxicity in the gastrointestinal tract. In particular, it was shown that adenovirus mediated expression of DKK1 in adult mice markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration, severe body weight loss and mortality from colitis and systemic infection. In particular, LRP5 and LRP6 are expressed in the intestine in the proliferative epithelial cells and are required for proliferation of the intestinal epithelium, suggesting that LRP5 and LRP6 inhibition may be toxic for this and other normal tissues (Zhong et al. “Lrp5 and Lrp6 play compensatory roles in mouse intestinal development”. J Cell Biochem. 2012; 113(1):31-8). This makes it doubtful whether agents which inhibit LRP5 and LRP6, or which inhibit the Wnt (Wnt1 and Wnt3a) signaling pathway in general, can be used for therapeutic purposes, e.g. can be developed as anti-cancer drugs.
WO2009/056634 refers to LRP6 binding molecules that may either interact with the Wnt1 signaling pathway or with the Wnt3/3a signaling pathway, which may be antagonistic or agonistic, and which may be used for diagnostic purposes or to treat “Wnt signaling-related disorders”, such as osteoarthritis, polycystic kidney disease, or cancer. No specific examples for such binding molecules, defined by their amino acid sequence, are provided in this document.
WO2011/138391 and WO2011/138392 are disclosing multivalent LRP6 binding antibodies. WO2011/138391 is claiming antibodies which are blocking one Wnt signaling pathway (Wnt1 or Wnt3) without potentiating the other pathway (Wnt3 or Wnt1, respectively). WO2011/138392 i.a. provides antibodies or antibody fragments which potentiate Wnt signaling by LRP6 receptor clustering.
WO2011/138391 explains that for achieving the desired effect, LRP6 binding molecules need to be formatted into full length IgG antibodies. Examples of LRP6 biparatopic molecules are provided which include an IgG molecule, having a first binding specificity, coupled to a single chain Fv portion, having the second binding specificity. Some formats are described as having significantly reduced thermal stability (Tm of 50 to 52° C.). An Fc portion may impart effector functions on an IgG molecule, such as complement-dependent cytotoxicity (CDC) or antibody-dependent cellular toxicity (ADCC).
WO2013/067355 discloses half-life extended biparatopic LRP6 binding scFv immunoglobulin constructs, derived from IgG molecules disclosed in WO2011/138391.
WO2011/119661 discloses antibodies that bind to LRP6 and inhibit the signaling induced by a first Wnt isoform, esp. by Wnt3 or Wnt3a, but potentiate signaling induced by a second Wnt isoform, which may be a Wnt1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a or 10b isoform. Bispecific molecules are disclosed which bind to the E1-E2 region of LRP6 as well as to the E3-E4 region of LRP6. The knob-in-hole technique was used to generate bispecific antibodies.
Identification of the binding epitopes (defined amino acid residues within the LRP6 ectodomain/beta-propeller regions) involved in the binding of the LRP6 antibodies is not provided in WO2009/056634, nor in WO2011/138391 or WO2013/067355, and only partially in WO2011/119661. In particular, LRP6 binding antibodies can inhibit Wnt signaling via alternative mechanisms according to binding to different regions of LRP6, including competing with Wnts directly or inhibiting formation of ternary receptor complexes (Wnt-LRP6-Frizzled), whereas others enhance signaling, possibly by receptor clustering (Ahn et al. “Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6”. Dev Cell. 2011; 21(5):862-73).
However, none of the binding molecules described in the art has so far been authorized by health authorities for the use as a medicament to treat any disease. Specifically, such use requires very specific binding properties, the right specificity, so that such molecules does or does not bind, activate or inhibit other targets (e.g. resulting in undesired activation or inhibition of other signaling pathways, or lack of activation or inhibition with respect to target isoforms), in the case of bi- or multispecific agents the right balance between the two or more binding specificities, suitable pharmacokinetic and -dynamic properties, an acceptable toxicological profile, and of course in vivo efficacy.
In view of the above, there is a need for novel therapeutic agents that allow an efficient treatment of several types of cancer diseases and tumors. It is thus an object of the invention to provide such pharmacologically active agents that can be used in the treatment of several cancer diseases, including NSCLC and TN BC.
In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods of treatment that provide certain advantages compared to the agents, compositions and/or methods currently used and/or known in the art. These advantages include in vivo efficacy, improved therapeutic and pharmacological properties, less side effects, and other advantageous properties such as improved ease of preparation or reduced costs of goods, especially as compared to candidate drugs already known in the art.